Biocatalysts, i.e., free or conjugated enzymes, offer unique advantages over classical chemical methods for producing a wide variety of products. In particular, biocatalysts are highly selective, i.e., able to differentiate between similar molecules or fragments; mild, i.e., minimize side reactions; and are often more environmentally friendly than classical chemical catalysts. However, all biocatalysts on the market either cannot be reused or do not work efficiently in heterogeneous reaction systems. Consequently, biocatalysis, i.e., methods of organic chemistry employing biocatalysts, is often overlooked for large-scale industrial applications.
Biocatalysts are currently commercially available in three forms: free enzymes, immobilized enzymes, and cross-linked enzyme crystal (CLEC(copyright); Altus, Cambridge, Mass.) biocatalysts. Free enzymes are soluble in aqueous reaction systems and act as any other homogeneous catalyst. Free enzymes work well in both homogeneous and heterogeneous reaction systems. However, problems associated with free enzymes include low stability under work-up conditions and significant surface activity of enzyme-containing solutions, especially at high concentrations. This makes the reuse of free enzymes practically impossible and complicates product purification for many large-scale industrial applications. Thus, most free enzymes are not cost-effective for large-scale industrial applications.
Immobilized enzymes are enzymes that are attached to a chemically inert solid carrier. Thus, immobilized enzymes are easier to separate from homogeneous reaction systems than free enzymes. However, most industrial processes involve heterogeneous reaction systems with insoluble starting materials or products or both. On the industrial scale, separating insoluble reaction products from insoluble immobilized enzymes is often the bottleneck in industrial processes and/or not cost-effective. Moreover, the activity of immobilized enzymes in heterogeneous reaction systems constitutes only a fraction of their activity in homogeneous reaction systems [Hayashi, T. and Ikada, Y., Biotechnol. Bioeng. 35:518 (1990)]. In addition, the active enzyme content in most commercially-available, covalently bound immobilized enzyme preparations is usually well below 1% by total weight (see Table 1).
Table 1 provides a comparison of the activities (U/g) of commercially-available free and immobilized enzyme preparations listed in their respective catalogs. The activities shown in Table 1 are corrected for protein content where that information is available in the same catalog for a given preparation. It was assumed that 100% of the protein in a given preparation is active enzyme. Comparisons between the activities of free and immobilized enzymes were made only where activity was measured using the same method. The Roche catalog in some cases gives lower limits of enzyme activity. Therefore, the calculated enzyme content of a particular immobilized enzyme preparation may represent a lower limit of its respective value. However, these data demonstrate that the actual amount of active enzyme in a particular immobilized enzyme preparation is quite low when compared to free enzyme preparations. As a result of the difficulty to reuse immobilized enzymes in heterogeneous reaction systems and the small percentages of active enzyme in commercially-available immobilized enzyme preparations, immobilized enzymes are less preferred than classical chemical techniques for many large-scale industrial applications.
CLEC(copyright) biocatalysts are fine enzyme crystals that are cross-linked and, therefore, insoluble in aqueous media. Because CLEC(copyright) biocatalysts are insoluble in aqueous media, CLEC(copyright) biocatalysts can be more easily separated from soluble starting materials and products than free enzymes. Further, because of their fine structure, CLEC(copyright) biocatalyst activity is close to the activity of free enzymes. However, CLEC(copyright) biocatalysts are expensive and possess most of the shortcomings of immobilized enzymes. Thus, a need exists for low-cost, reusable industrial enzyme biocatalysts that are able to work in heterogeneous reaction systems with the same activity as free enzymes.
Too fill this void, a few attempts have been made to design reversibly soluble enzyme-biocatalysts. These biocatalysts are reversibly soluble dependent upon minor changes in the reaction environment, such as temperature, salt concentration, pH, etc. Thus, when in the soluble state, reversibly soluble enzyme biocatalysts are able to function effectively in heterogeneous reaction systems. Further, the reversibly soluble nature of these biocatalysts permits precipitation recovery and reuse. Thus, reversibly soluble enzyme biocatalysts overcome the disadvantages of free enzymes, immobilized enzymes and CLEC(copyright) biocatalysts with respect to reuse and the ability to function in heterogeneous reaction systems. In all cases, reversibly soluble enzyme biocatalysts are conjugates of an enzyme with a reversibly soluble polymer i.e., reversibly soluble enzyme-polymer conjugates.
Reversibly soluble enzyme-polymer conjugates have been made with the following enzymes: chymotrypsin [Chen, J.-P. and Hsu, M.-S., J. Molec. Catalysis B: Enzymatic 2:233 (1997)], trypsin [Shiroya, T., Yasui, M., Fujimoto, K. and Kawaguchi, H., Colloid Surfaces B: Biointerfaces, 4:275 (1995)], xcex2-D-glucosidase [Chen, G. and Hoffman, A. S., J. Biomater. Sci. Polym. Edn., 5:371 (1994)], lactate dehydrogenase [Galaev, I. Yu. and Mattiasson, B, Biotechnol. Bioeng., 41:1101 (1993)], thermnolysin [Liu, F., Tao, G. and Zhuo, R., Polymer J., 25:561 (1993)] and lipase [Takeuchi, S., Omodaka, I., Hasegawa, K., Maeda, Y. and Kitano, Y., Makromol. Chem., 194:1991 (1993)]. However, current methods for producing reversibly soluble enzyme-polymer conjugates produce biocatalysts with enzyme activities, on a weight basis, that are usually significantly lower than those of free enzymes.
Significant loss of the enzyme during binding is one of the main shortcomings of current methods for producing enzyme-polymer conjugates. For example, the binding efficiency of a current method for conjugating chymotrypsin to a copolymer of N-iso-propylacrylamide (NIPAAM) and N-acrylosuccinimide is only 30-40% [Chen, J.-P. and Hsu, M.-S., J. Molec. Catalysis B: Enzymatic, 2: 233 (1997)]. When conjugating thermolysin to the same copolymer, the binding efficiency is about twice as low as with chymotrypsin [Liu, F., Tao, G. and Zhuo, R., Polymer J., 25: 561 (1993)]. Only 43% of enzyme binds with the polymer in a reaction between thermolysin and a NIPAAM-based copolymer containing oxirane groups [Vorlop, K.-D., Steinke, K., Wullbrandt, D., Schlingmann, M., U.S. Pat. No. 5,310,786 (1994)]. Binding of chymotrypsin to a polymerized liposome gives much better results, i.e., between 36% and 89% of the enzyme was coupled [Suh, Y., Jin, X,-H., Dong, X.-Y., Yu, K. and Zhou, X. Z., Appl. Biochem. Biotechnol., 56: 331 (1996)], but the procedure is quite cumbersome. When enzyme-polymer conjugates were prepared by carbodiimide-assisted coupling of trypsin with carboxylated poly-(NIPAAM), only a small percentage of the total amount of trypsin was bound to the polymer [Chen, G. and Hoffman, A. S., J. Biomater. Sci. Polym. Edn., 5: 371-382 (1994)].
Many enzymes are quite expensive and, from an industrial standpoint, the degree of enzyme incorporation into a polymer is the key parameter affecting cost-effectiveness of the preparation of reversibly soluble enzyme-polymer conjugates. Therefore, current methods for producing reversibly soluble enzyme-polymer conjugates are not cost-effective for large-scale industrial applications. Moreover, while the concept of reversibly soluble enzyme-polymer conjugates catalyzing reactions with insoluble substrates has been demonstrated, there are no examples of their use with insoluble or poorly soluble substrates and/or products of commercial value in which the reversibly soluble enzyme-polymer conjugates could reveal their full potential. No optimization of current methods for producing enzyme-polymer conjugates has been performed. All current methods for producing enzyme-polymer conjugates are hit-and-miss methods and their performance is far from their potential.
In one aspect, present invention is directed to a method for producing reversibly soluble, catalytically active enzyme-polymer conjugates by incorporating enzymes modified to contain free vinyl double bonds into reversibly soluble polymers (xe2x80x9csmartxe2x80x9d polymers) during polymerization. Smart polymers precipitate following a slight change in environmental conditions. Thus, when modified enzymes are incorporated into smart polymers, the biocatalysts obtained can be precipitated without destroying the delicate enzyme(s). Later the biocatalyst can be solubilized again at the initial environmental conditions. Therefore, reversibly soluble biocatalysts acquire the advantages of free enzymes (high activity in heterogeneous systems) and those of immobilized enzymes and CLEC(copyright) biocatalysts (easy work-up and the possibility of the reuse). In particular, the present method is directed to a process for producing a reversibly soluble, catalytically active enzyme-polymer conjugate comprising:
(a) contacting a free enzyme having at least one free amino group with a modifying agent, the modifying agent having at least one vinyl double bond and an active acylating group, the active group forming an amide bond between the modifying agent and the free amino group of the enzyme and producing a modified enzyme having a free vinyl double bond;
(b) recovering the modified enzyme;
(c) contacting the modified enzyme with a soluble monomer having the structure R1R2CCR3R4, wherein R1 is selected from hydrogen, carboxy or phenyl moieties, R2 is a hydrogen moiety; R3 is selected from hydrogen, methyl, carboxy, sulfo, or 2-pyridine moieties and R4 is selected from hydrogen, methyl, methoxy, aminopropyl, N,N-dimethylaminopropyl, N N-diethylaminopropyl, 
xe2x80x83or C(O)NR5R6, wherein R5 is selected from hydrogen, methyl, ethyl, propyl, iso-propyl, or propyl, tetrahydro-pyran-2-yl, 2-methoxy-ethyl, R6 is selected from ethyl, propyl, iso-propyl, cyclo-propyl, tetrahydropyrano-2-yl, 2-methoxy-ethyl, 3-methoxy-propyl, 3-iso-propoxy-propyl or xe2x80x94C(CH3)2xe2x80x94C(O)xe2x80x94NHR7, wherein R7 is selected from methyl, ethyl, iso-propyl, 3-methoxy-propyl or 2,2-diethoxy-ethyl, and wherein the monomer is dissolved in a solution and is selected from a group of monomers which when polymerized contain both hydrophobic and hydrophilic regions; and
(d) contacting an initiating agent or agents with the monomer, thereby causing a bond to form between the modified enzyme and the monomer and producing an active enzyme-polymer conjugate that reversibly changes its solubility upon a change in the temperature, salt concentration or pH of the solution.
In another aspect, the invention is directed to a reversibly soluble, catalytically active enzyme-polymer conjugate product.